Process for the preparation of D-pantothenic acid and/or salts thereof

ABSTRACT

A process for the preparation of D-pantothenic acid and/or salts thereof or feedstuffs additives comprising these by fermentation of microorganisms of the Enterobacteriaceae family, in particular those which already produce D-pantothenic acid, in which the nucleotide sequence(s) in the microorganisms which code(s) for the pepB gene is/are enhanced, in particular over-expressed.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

[0001] The present invention relates to a process for the preparation ofD-pantothenic acid and salts thereof or mixtures comprising thesecompounds using microorganisms of the Enterobacteriaceae family in whichat least the pepB gene is enhanced.

DESCRIPTION OF THE BACKGROUND

[0002] Pantothenic acid is produced worldwide in an order of magnitudeof several thousand tons a year. It is used inter alia in humanmedicine, in the pharmaceuticals industry and in the foodstuffsindustry. A large portion of the pantothenic acid produced is used fornutrition of stock animals such as poultry and pigs.

[0003] Pantothenic acid can be prepared by chemical synthesis, orbiotechnologically by fermentation of suitable microorganisms insuitable nutrient solutions. In the chemical synthesis, DL-pantolactoneis an important precursor. It is prepared in a multi-stage process fromformaldehyde, isobutylaldehyde and cyanide, and in further processsteps, the racemic mixture is separated, D-pantolactone is subjected toa condensation reaction with β-alanine, and D-pantothenic acid isobtained in this way.

[0004] The typical commercial form is the calcium salt of D-pantothenicacid. The calcium salt of the racemic mixture of D,L-pantothenic acid isalso customary.

[0005] The advantage of the fermentative preparation by microorganismslies in the direct formation of the desired stereoisomeric form, that isto say the D-form, which is free from L-pantothernic acid.

[0006] Various types of bacteria, such as e.g. Escherichia coli (E.coli), Arthrobacter ureafaciens, Corynebacterium erythrogenes,Brevibacterium ammoniagenes, and also yeasts, such as e.g. Debaromycescastellii, can produce D-pantothenic acid in a nutrient solution whichcomprises glucose, DL-pantoic acid and β-alanine, as shown in EP-A 0 493060. EP-A 0 493 060 furthermore shows that in the case of E. coli, theformation of D-pantothenic acid is improved by amplification ofpantothenic acid biosynthesis genes from E. coli which are contained onthe plasmids pFV3 and pFV5 in a nutrient solution comprising glucose,DL-pantoic acid and β-alanine.

[0007] EP-A 0 590 857 and U.S. Pat. No. 5,518,906 describe mutantsderived from E. coli strain IFO3547, such as FV5714, FV525, FV814,FV521, FV221, FV6051 and FV5069, which carry resistances to variousantimetabolites, such as salicylic acid, α-ketobutyric acid,β-hydroxyaspartic acid, O-methylthreonine and α-ketoisovaleric acid.They produce pantoic acid in a nutrient solution comprising glucose, andD-pantothenic acid in a nutrient solution comprising glucose andβ-alanine. It is furthermore stated in EP-A 0 590 857 and U.S. Pat. No.5,518,906 that after amplification of the pantothenic acid biosynthesisgenes panB, panC and panD, which are said to be contained on the plasmidpFV31, in the above-mentioned strains the production of D-pantoic acidin nutrient solutions comprising glucose and the production ofD-pantothenic acid in a nutrient solution comprising glucose andβ-alanine is improved.

[0008] WO 97/10340 furthermore reports on the favorable effect of theenhancement of the ilvGM operon on the production of D-pantothenic acid.Finally, EP-A-1001027 reports on the effect of the enhancement of thepanE gene on the formation of D-pantothenic acid.

[0009] According to known procedures, the D-pantothenic acid or thecorresponding salt is isolated from the fermentation broth and purified(EP-A-0590857 and WO 96/33283) and used accordingly in purified form, orthe fermentation broth comprising D-pantothenic acid is dried in total(EP-A-1050219) and used in particular as a feedstuffs additive.

SUMMARY OF THE INVENTION

[0010] It is an object of the invention to provide new methods forimproved fermentative preparation of D-pantothenic acid and/or saltsthereof, and animal feedstuffs additives comprising these.

[0011] The invention provides a process for the preparation ofD-pantothenic acid and/or salts thereof using microorganisms of theEnterobacteriaceae family which in particular already produceD-pantothenic acid and in which at least one, preferably endogenousnucleotide sequence(s) which code(s) for the pepB gene is enhanced, inparticular over-expressed.

[0012] In particular, the process is a process which is characterized inthat the following steps are carried out:

[0013] a) fermentation of microorganisms of the Enterobacteriaceaefamily which produce D-pantothenic acid and in which at least the pepBgene is enhanced, in particular over-expressed; the gene which codes forpeptidase B and optionally alleles of this gene-are enhanced, inparticular over-expressed, under conditions suitable for the formationof the gene product; further genes of the pantothenic acid biosynthesispathway are optionally attenuated or enhanced at the same time in orderto increase the production of pantothenic acid;

[0014] b) the fermentation is optionally carried out in the presence ofalkaline earth metal compounds, these being added to the fermentationbroth continuously or discontinuously in preferably stoichiometricamounts;

[0015] c) concentration of the D-pantothenic acid or the correspondingsalts in the medium or the fermentation broth or optionally in the cellsof the microorganisms of the Enterobacteriaceae family; and

[0016] d) after conclusion of the fermentation, isolation of theD-pantothenic acid, and/or of the corresponding salt(s).

[0017] The invention also provides a process in which, after conclusionof the fermentation, some or all (≧0 to 100%) of the biomass remains inthe fermentation broth, and the broth obtained in this way is processed,optionally after concentration, to a solid mixture which comprisesD-pantothenic acid and/or salts thereof and preferably comprises furtherconstituents from the fermentation broth.

[0018] These further constituents are, above all, the dissolvedcompounds which originate from the feed medium and soluble organiccompounds which are formed.

[0019] A more complete appreciation of the invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following Figures inconjunction with the detailed description below.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1: Map of the plasmid pTrc99A-pepB containing the pepB gene.

[0021] The length data are to be understood as approx. data. Theabbreviations and designations used have the following meaning: Amp:Ampicillin resistance gene lacI: Gene for the repressor protein of thetrc promoter Ptrc: trc promoter region, IPTG-inducible pepB: Codingregion of the pepB gene 5S: 5S rRNA region rrnBT: rRNA terminator regionbps Base pairs

[0022] The abbreviations for the restriction enzymes have the followingmeaning:

[0023] BamHI: Restriction endonuclease from Bacillus amyloliquefaciens

[0024] EcoRV: Restriction endonuclease from Escherichia coli B946

[0025] PvuI: Restriction endonuclease from Proteus vulgaris

[0026] SalI: Restriction endonuclease from Streptomyces albus

DETAILED DESCRIPTION OF THE INVENTION

[0027] When D-pantothenic acid or pantothenic acid or pantothenate arementioned in the following text, this means not only the free acids butalso the salts of D-pantothenic acid, such as e.g. the calcium, sodium,ammonium or potassium salt.

[0028] “Endogenous genes” or “endogenous nucleotide sequences” areunderstood as meaning the genes or nucleotide sequences present in thepopulation of a species.

[0029] The term “enhancement” in this connection describes the increasein the intracellular activity of one or more enzymes or proteins in amicroorganism which are coded by the corresponding DNA, for example byincreasing the number of copies of the gene or genes, of the ORF (OpenReading Frame) or ORFs, using a potent promoter or a gene or allele orORF which codes for a corresponding enzyme or protein with a highactivity, and optionally combining these measures.

[0030] By enhancement measures, in particular over-expression, theactivity or concentration of the corresponding enzyme or protein is ingeneral increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%,300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on that ofthe wild-type protein or wild-type enzyme or the activity orconcentration of the protein or enzyme in the starting microorganism.

[0031] The microorganisms which the present invention provides canproduce D- pantothenic acid from glucose, sucrose, lactose, fructose,maltose, molasses, starch, cellulose or from glycerol and ethanol. Theyare representatives of Enterobacteriaceae, in particular of the genusEseherichia. Of the genus Escherichia, the specie Escherichia coli is tobe mentioned in particular. Within the species Escherichia coli theso-called K-12 strains, such as e. g. the strains MG1655 or W3110(Neidhard et al.: Escherichia coli and Salnonella. Cellular andMolecular Biology (ASM Press, Washington D.C.)) or the Escherichia coliwild type strain IFO3547 (Institute of Fermentation, Osaka, Japan) andmutants derived from these which have the ability to produceD-pantothenic acid are suitable.

[0032] Suitable D-pantothenic acid-producing strains of the genusEscherichia, in particular of the species Escherichia coli, are, forexample

[0033]Escherichia coli FV5069/pFV31

[0034]Escherichia coli FV5069/pFV202

[0035]Escherichia coli FE6/pFE80 and

[0036]Escherichia coli KE3

[0037] It has been found that Enterobacteriaceae produce D-pantothenicacid in an improved manner after enhancement, in particularover-expression of the pepB gene. The use of endogenous genes ispreferred.

[0038] The nucleotide sequences of the genes or open reading frames(ORF) of Escherichia coli are known, and can also be found in the genomesequence of Escherichia coli published by Blattner et al. (Science 277,1453-1462 (1997)).

[0039] The following information, inter alia, on the pepB gene can befound in the following: Description: Peptidase B Reference: Hermsdorf etal., International Journal of Peptide and Protein Research 13:146-51(1979); Suzuki et al. Journal of Fermentation and Bioengineering82:392-397 (1996) Accession No.: AE000339

[0040] The gene described in the reference cited above can be usedaccording to the invention. Alleles of the gene or open reading frameswhich result from the degeneracy of the genetic code or due to sensemutations of neutral function can furthermore be used, the activity ofthe proteins being substantially unchanged.

[0041] To achieve an over-expression, the number of copies of thecorresponding genes can be increased, or the promoter and regulationregion or the ribosome binding site upstream of the structural gene canbe mutated. Expression cassettes which are incorporated upstream of thestructural gene act in the same way. By inducible promoters, it isadditionally possible to increase the expression in the course offermentative D-pantothenic acid production. The expression is likewiseimproved by measures to prolong the life of the m-RNA. Furthermore, theenzyme activity is also increased by preventing the degradation of theenzyme protein. The genes or gene constructs can either be present inplasmids with a varying number of copies, or can be integrated andamplified in the chromosome. Alternatively, an over-expression of thegenes in question can furthermore be achieved by changing thecomposition of the media and the culture procedure.

[0042] Instructions in this context can be found by one skilled in theart, inter alia, in Chang and Cohen (Journal of Bacteriology134:1141-1156 (1978)), in Hartley and Gregori (Gene 13:347-353 (1981)),in Amann and Brosius (Gene 40:183-190 (1985)), in de Broer et al.(Proceedings of the National Academy of Sciences of the United States ofAmerica 80:21-25 (1983)), in LaVallie et al. (BIO/TECHNOLOGY 11, 187-193(1993)), in PCT/US97/13359, in Llosa et al. (Plasmid 26:222-224 (1991)),in Quandt and Klipp (Gene 80:161-169 (1989)), in Hamilton (Journal ofBacteriology 171:4617-4622 (1989), in Jensen and Hammer (Biotechnologyand Bioengineering 58, 191-195 (1998) and in known textbooks of geneticsand molecular biology.

[0043] Plasmid vectors which are capable of replication inEnterobacteriaceae, such as e.g. cloning vectors derived from pACYC184(Bartolomé et al.; Gene 102, 75-78 (1991)), pTrc99A (Amann et al.; (Gene69:301-315 (1988)) or pSC101 derivatives (Vocke and 20 Bastia,Proceedings of the National Academy of Science USA 80 (21):6557-6561(1983)) can be used. A strain transformed with one or more plasmidvectors where the plasmid vector(s) carries at least one nucleotidesequence which codes for the pepB gene can be employed in a processaccording to the invention.

[0044] It may furthermore be advantageous for the production ofD-pantothenic acid with strains of the Enterobacteriaceae family, inaddition to the enhancement of the pepB gene, for one or more of thegenes chosen from the group consisting of

[0045] the ilvGM operon which codes for acetohydroxy-acid synthase II(WO 97/10340),

[0046] the panB gene which codes for ketopantoate hydroxymethyltransferase (U.S. Pat. No. 5,518,906),

[0047] the pane gene which codes for ketopantoate reductase(EP-A-1001027),

[0048] the panD gene which codes for aspartate decarboxylase (U.S. Pat.No 5,518,906),

[0049] the panC gene which codes for pantothenate synthetase (U.S. Pat.No 5,518,906),

[0050] the glyA gene which codes for serine hydroxymethyl transferase(Plamann et al., Nucleic Acids Research 11(7):2065-2075(1983)),

[0051] the genes gcvT, gcvH and gcvP which code for the glycine cleavagesystem (Okamura-Ikeda et al., European Journal of Biochemistry 216,539-548 (1993)),

[0052] the serA gene which codes for phosphoglyceric acid dehydrogenase(Tobey und Grant, Journal of Biological Chemistry261:12179-12183(1986)),

[0053] the serA(FBR) allele which codes for “feed back” resistantvariants of phosphoglyceric acid dehydrogenase (DE-A-4232468),

[0054] the serC gene which codes for phosphoserine transaminase (Duncanund Coggins, Biochemical Journal 234:49-57 (1986)),

[0055] the bfr gene which codes for bacterioferrin (Andrews et al.,Journal of Bacteriology 171:3940-3947 (1989)),

[0056] the hns gene which codes for the DNA-binding protein HLP-II(reference: Pon et al., Molecular and General Genetics 212:199-202(1988)),

[0057] the pgm gene which codes for phosphoglucomutase (Lu and Kleckner,Journal of Bacteriology 176:5847-5851 (1994)),

[0058] the mdh gene which codes for malate dehydrogenase (Sutherland undMcAlister-Henn, Journal of Bacteriology 1985 163:1074-1079 (1985)),

[0059] the cysK gene which codes for cysteine synthase A (Boronat etal., Journal of General Microbiology 130:673-685 (1984)),

[0060] the fda gene which codes for fructose bisphosphate aldolase(class II) (Alefounder et al., Biochemical Journal 257:529-534 (1989)),

[0061] the dldH gene which codes for NADH-dependent lipoamidedehydrogenase (reference: Stephens et al., European Journal ofBiochemistry 135:519-527 (1983)),

[0062] the aldH gene which codes for NADP-dependent aldehydedehydrogenase (Heim and Strehler, Gene 99:15-23 (1991)) and

[0063] the adk gene which codes for adenylate kinase (Brune et al.,Nucleic Acids Research 13:7139-7151 (1985))

[0064] to be enhanced, in particular over-expressed, individually ortogether. The use of endogenous genes is preferred.

[0065] Finally, it may be advantageous for the production ofD-pantothenic acid with strains of the Enterobacteriaceae family, inaddition to the enhancement of the pepB gene, for one or more of thegenes chosen from the group consisting of

[0066] the avtA gene which codes for transaminase C (EP-A-1001027)

[0067] the poxB gene which codes for pyruvate oxidase (Grabau andCronan, Nucleic Acids Research. 14 (13), 5449-5460 (1986))

[0068] the pckA gene which codes for PEP carboxykinase (Medina et al.,Journal of Bacteriology 172, 7151-7156 (1990))

[0069] to be attenuated, in particular eliminated or expressed at a lowlevel, individually or together.

[0070] The term “attenuation” in this connection describes the reductionor elimination of the intracellular activity of one or more enzymes orproteins in a microorganism which are coded by the corresponding DNA,for example by using a weak promoter or using a gene or allele whichcodes for a corresponding enzyme or protein with a low activity orinactivates the corresponding gene or enzyme (protein), and optionallycombining these measures.

[0071] By attenuation measures, including reduction in expression, theactivity or concentration of the corresponding protein is in generalreduced to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of theactivity or concentration of the wild-type protein or of the activity orconcentration of the protein in the starting microorganism.

[0072] In addition to over-expression of the pepB gene it mayfurthermore be advantageous for the production of D-pantothenic acid toeliminate undesirable side reactions (Nakayama: “Breeding of Amino AcidProducing Microorganisms”, in: Overproduction of Microbial Products,Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).Bacteria in which the metabolic pathways which reduce the formation ofD-pantothenic acid are at least partly eliminated can be employed in theprocess according to the invention.

[0073] The microorganisms produced according to the invention can becultured in the batch process (batch culture), the fed batch (feedprocess) or the repeated fed batch process (repetitive feed process). Asummary of known culture methods is described in the textbook by Chmiel(Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik [BioprocessTechnology 1. Introduction to Bioprocess Technology (Gustav FischerVerlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktorenund periphere Einrichtungen [Bioreactors and Peripheral Equipment](Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

[0074] The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981). Sugars and carbohydrates,such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses,starch and cellulose, oils and fats, such as e.g. soya oil, sunfloweroil, groundnut oil and coconut fat, fatty acids, such as e.g. palmiticacid, stearic acid and linoleic acid, alcohols, such as e.g. glyceroland ethanol, and organic acids, such as e.g. acetic acid, can be used asthe source of carbon. These substances can be used individually or as amixture.

[0075] Organic nitrogen-containing compounds, such as peptones, yeastextract, meat extract, malt extract, corn steep liquor, soya bean flourand urea, or inorganic compounds, such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,can be used as the source of nitrogen. The sources of nitrogen can beused individually or as a mixture.

[0076] Phosphoric acid, potassium dihydrogen phosphate or dipotassiumhydrogen phosphate or the corresponding sodium-containing salts can beused as the source of phosphorus. The culture medium must furthermorecomprise salts of metals, such as e.g. magnesium sulfate or ironsulfate, which are necessary for growth. Finally, essential growthsubstances, such as amino acids and vitamins, can be employed inaddition to the above-mentioned substances. Precursors of pantothenicacid, such as aspartate, β-alanine, ketoisovalerate, ketopantoic acid orpantoic acid and optionally salts thereof, can moreover be added to theculture medium. The starting substances mentioned can be added to theculture in the form of a single batch, or can be fed in during theculture in a suitable manner.

[0077] Basic compounds, such as sodium hydroxide, potassium hydroxide,ammonia or aqueous ammonia, or acid compounds, such as phosphoric acidor sulfuric acid, can be employed in a suitable manner to control the pHof the culture.

[0078] For the preparation of alkaline earth metal salts of pantothenicacid, in particular the calcium salt or magnesium salt, it is equallypossible to add the suspension or solution of an inorganic compoundcontaining an alkaline earth metal, such as, for example, calciumhydroxide or MgO, or of an organic compound, such as the alkaline earthmetal salt of an organic acid, for example calcium acetate, continuouslyor discontinuously during the fermentation. For this purpose, the cationnecessary for preparation of the desired alkaline earth metal salt ofD-pantothenic acid is introduced into the fermentation broth directly inthe desired amount, preferably in an amount of 0.95 to 1.1 equivalents.

[0079] However, the salts can also be formed after conclusion of thefermentation by addition of the inorganic or organic compounds to thefermentation broth, from which the biomass has optionally been removedbeforehand.

[0080] Antifoams, such as e.g. fatty acid polyglycol esters, can beemployed to control the development of foam. Suitable substances havinga selective action, e.g. antibiotics, can be added to the medium tomaintain the stability of plasmids. To maintain aerobic conditions,oxygen or oxygen-containing gas mixtures, such as e.g. air, areintroduced into the culture. The temperature of the culture is usually25° C. to 45° C, and preferably 30° C. to 40° C. The pH is in generalbetween 5.0 to 8.0, preferably 5.5 to 7.6. The fermentation is continueduntil a maximum of D-pantothenic acid has formed. This target is usuallyreached within 10 hours to 160 hours.

[0081] The D-pantothenic acid or the corresponding salts ofD-pantothenic acid contained in the fermentation broth can then beisolated and purified in accordance with known procedures.

[0082] It is also possible for the fermentation broths comprisingD-pantothenic acid and/or salts thereof preferably first to be freedfrom all or some of the biomass by known separation methods, such as,for example, centrifugation, filtration, decanting or a combinationthereof. However, it is also possible to leave the biomass in itsentirety in the fermentation broth. In general, the suspension orsolution is preferably concentrated and then worked up to a powder, forexample with the aid of a spray dryer or a freeze-drying unit. Thispowder is then in general converted by suitable compacting orgranulating processes, e.g. also build-up granulation, into acoarser-grained, free-flowing, storable and largely dust-free productwith a particle size distribution of preferably 20 to 2000 μm, inparticular 100 to 1400 μm. In the granulation or compacting it isadvantageous to employ conventional organic or inorganic auxiliarysubstances or carriers, such as starch, gelatin, cellulose derivativesor similar substances, such as are conventionally used as binders,gelling agents or thickeners in foodstuffs or feedstuffs processing, orfurther substances, such as, for example, silicas, silicates orstearates.

[0083] Alternatively, the fermentation product, with or without furtherof the conventional fermentation constituents, can be absorbed, inparticular sprayed, on to an organic or inorganic carrier substancewhich is known and conventional in feedstuffs processing, such as, forexample, silicas, silicates, grits, brans, meals, starches, sugars orothers, and/or stabilized with conventional thickeners or binders. Useexamples and processes in this context are described in the literature(Die Mühle+Mischfuttertechnik 132 (1995) 49, page 817).

[0084] These mixtures comprising the carrier substances can also beprocessed to a product with the desired particle size distribution bygranulation processes.

[0085] D-Pantothenic acid and/or the desired salt of D-pantothenic acidor a formulation comprising these compounds is optionally added in asuitable process stage during or after the fermentation in order toachieve or establish the content of pantothenic acid desired in theproduct or the desired salt.

[0086] The desired content of pantothenic acid and/or the desired saltis in general in the range from 20 to 80 wt.% (based on the dry weight).

[0087] The concentration of pantothenic acid can be determined withknown chemical (Velisek; Chromatographic Science 60, 515-560 (1992)) ormicrobiological methods, such as e.g. the Lactobacillus plantarum test(DIFCO MANUAL, 10^(th) Edition, p. 1100-1102; Michigan, USA).

[0088] The present invention is explained in more detail in thefollowing with the aid of embodiment examples.

[0089] The minimal (M9) and complete media (LB) for Escherichia coliused are described by J. H. Miller (A Short Course in Bacterial Genetics(1992), Cold Spring Harbor Laboratory Press). The isolation of plasmidDNA from Escherichia coli and all techniques of restriction, ligation,Klenow and alkaline phosphatase treatment are carried out by the methodof Sambrook et al. (Molecular cloning—A laboratory manual (1989), ColdSpring Harbor Laboratory Press). The transformation of Escherichia coliis carried out by the method of Chung et al. (Proceedings of theNational Academy of Sciences of the United States of America (1989) 86:2172-2175) or by the method of Chuang et. al. (Nucleic Acids Research(1995) 23: 1641).

EXAMPLES

[0090] Having generally described this invention, a furtherunderstanding can be obtained by reference to certain specific exampleswhich are provided herein for purposes of illustration only and are notintended to be limiting unless otherwise specified.

Example 1 Construction of the Expression Plasmid pTrc99A-pepB

[0091] The pepB gene from E. coli K12 is amplified using the polymerasechain reaction (PCR) and synthetic oligonucleotides. Starting from thenucleotide sequence of the pepB gene in E. coli K12 MG1655 (AccessionNumber AE000339, Blattner et al. (Science 277, 1453-1462 (1997)), PCRprimers are synthesized (MWG Biotech, Ebersberg, Germany). The 5′ endsof the primers are lengthened with recognition sequences for restrictionenzymes and two to four additional bases. This part of the primer isidentified in the following description by a hyphen (-). The recognitionsequence for BamHI is chosen for the 5′ primer and the recognitionsequence for SalI for the 3′ primer, which are marked by underlining inthe nucleotide sequence shown below: (SEQ ID No.1) Primer pepB5′:5′-CGCGGATCC-AACTGGCGGCCCTTT-3′ (SEQ ID No.2) Primer pepB3′: 5′-ACGCGTCGAC-CTGATGCGCTACGCT-3′

[0092] The chromosomal E. coli K12 MG1655 DNA employed for the PCR isisolated according to the manufacturer's instructions with “QiagenGenomic-tips 100/G” (QIAGEN, Hilden, Germany). A DNA fragment approx.800 bp in size can be amplified with the specific primers under standardPCR conditions (Innis et al. (1990) PCR Protocols. A guide to methodsand applications, Academic Press) with Pfu-DNA polymerase (PromegaCorporation, Madison, USA). The PCR product is ligated according to themanufacturer's instructions with the vector pCR-Blunt II-TOPO (ZeroBlunt TOPO PCR Cloning Kit, Invitrogen, Groningen, The Netherlands) andtransformed into the E. coli strain TOP10. Selection of plasmid-carryingcells takes place on LB agar, to which 50 μg/ml kanamycin are added.After isolation of the plasmid DNA, the vector pCR-Blunt II-TOPO-pepB iscleaved with the restriction enzymes BamHI and SalI and, afterseparation in 0.8% agarose gel, the pepB fragment is isolated with theaid of the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). Thevector pTrc99A (Amersham Biosciences, Freiburg, Germany) is cleaved withthe enzymes BamHI and SalI, subsequently dephosphorylated with alkalinephosphatase according to the manufacturer's instructions (AmershamBiosciences, Freiburg, Germany) and ligated with the pepB fragmentisolated. The E. coli strain XL1-Blue MRF′ (Stratagene, La Jolla, USA)is transformed with the ligation batch and plasmid-carrying cells areselected on LB agar, to which 50 μg/ml ampicillin is added. Successfulcloning can be demonstrated after plasmid DNA isolation by controlcleavage with the enzymes BamHI, SalI, EcoRV and PvuI. The plasmid iscalled pTrc99A-pepB (FIG. 1).

Example 2 Preparation of the Strains FE6-1/pTrc99A andFE6-1/pTrc99A-pepB

[0093] The E. coli strain FE6 is a valine-resistant mutant of E. coliK12 MG1655 (U.S. Pat. No. 6,171,845) and is deposited as DSM12379 at theDeutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ=GermanCollection of Microorganisms and Cell Cultures, Braunschweig, Germany).Starting from FE6, after incubation at 37° C. on minimal agar, to which2 g/L glucose and 1 g/L β-hydroxyaspartic acid are added, spontaneousmutants are isolated. A selected β-hydroxyaspartic acid-resistantindividual colony is then incubated on minimal agar, which comprises 2g/L glucose and 0.2 g/L O-methylthreonine, at 37° C. After this step, amutant called FE6-1 is resistant to L-valines, α-ketoisovaleric acid,β-hydroxyaspartic acid and O-methylthreonine. A pure culture of thestrain FE6-1 was deposited on Sep. 8, 2000 as DSM13721 at the DeutscheSammlung für Mikroorganismen und Zellkulturen (DSMZ=German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany).

[0094] The plasmids pTrc99A and pTrc99A-pepB are transformedindividually into the strain FE6-1 and plasmid-carrying cells areselected on LB agar, to which 50 μg/ml ampicillin are added. The strainsobtained are called FE6-1/pTrc99A and FE6-1/pTrc99A-pepB.

Example 3 Preparation of D-pantothenic Acid with Strains Derived fromFE6-1

[0095] The pantothenate production of the E. coli strains FE6-1/pTrc99Aand FE6-1/pTrc99A-pepB is checked in batch cultures of 10 ml containedin 100 ml conical flasks. For this, 10 ml of preculture medium of thefollowing composition: 2 g/l yeast extract, 10 g/l (NH₄)₂SO₄, 1 g/lKH₂PO₄, 0,5 g/l MgSO₄.7H₂O, 15 g/l CaCO₃, 20 g/l gl ampicillin areinoculated with an individual colony and incubated for 20 hours at 33°C. and 200 rpm on an ESR incubator from Kuihner AG (Birsfelden,Switzerland). In each case 200 μl of this preculture are transinoculatedinto 10 ml of production medium (25 g/l (NH₄)₂SO₄, 2 g/l KH₂PO₄, 1 g/lMgSO₄.7H₂O, 0.03 g/l FeSO₄.7H₂O, 0.018 g/l MnSO₄. 1H₂O, 30 g/l CaCO₃, 20g/l glucose, 20 g/l β-alanine, 250 mg/l thiamine) and the batch isincubated for 48 hours at 37° C. After the incubation the opticaldensity (OD) of the culture suspension is determined with an LP2Wphotometer from Dr. Lange (Düisseldorf, Germany) at a measurementwavelength of 660 nmn.

[0096] The concentration of the D-pantothenate formed is then determinedin the culture supernatant centrifuged off by means of High PerformanceLiquid Chromatography [column: Reversed Phase MZ-Aqua Perfect (diameter4,6 mm), mobile Phase 25 mM acetate buffer with 10% methanol, flow rate1 ml/min, RI detector].

[0097] The result of the experiment is shown in table 1. TABLE 1 ODPantothenate Strain (660 nm) mg/l FE6-1/pTrc99A 8.7 47FE6-1/pTrc99A-pepB 9.4 74

[0098] The publications cited in the Detailed Description of theInvention and the Examples above are incorporated herein by reference.

[0099] Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

[0100] This application is based on German Patent Application Serial No.101 28 780.1, filed on Jun. 13, 2001, and incorporated herein byreference.

1 6 1 27 DNA Artificial sequence Synthetic oligonucleotide 1 cggaattcgatcatggagtc agtcgag 27 2 27 DNA Artificial sequence Syntheticoligonucleotide 2 gcctctagag tcccatctac ggttggg 27 3 22 DNA Artificialsequence Synthetic oligonucleotide 3 ggtagcacca ctgcagagct tc 22 4 21DNA Artificial sequence Synthetic oligonucleotide 4 ggtcacaagggcgtctccaa g 21 5 20 DNA Artificial sequence Synthetic oligonucleotide 5accacagtcc atcggatcac 20 6 20 DNA Artificial sequence Syntheticoligonucleotide 6 tccaccaccc tgttgctgta 20

1. A process for the preparation of D-pantothenic acid and/or a saltthereof or a feedstuffs additive comprising this/these compound(s) byfermentation of microorganisms of the Enterobacteriaceae family, inwhich at least the nucleotide sequence(s) which code(s) for the pepBgene is/are enhanced, under conditions suitable for the formation of thepepB gene product peptidase B.
 2. The process according to claim 1,wherein the microorganism naturally produces D-pantothenic acid.
 3. Theprocess according to claim 1, wherein the nucleotide sequence(s) whichcode(s) for the pepB gene is/are over-expressed.
 4. The processaccording to claim 1, wherein the microorganism is transformed with atleast one plasmid which carries the pepB gene.
 5. The process accordingto claim 4, wherein the pepB gene is integrated into the chromosome inthe transformed microorganism.
 6. The process according to claim 1,wherein in the microorganism the promoter and regulation region upstreamof the structural gene is mutated to achieve the enhancement.
 7. Theprocess according to claim 1, wherein in the microorganism at least oneexpression cassette is incorporated upstream of the structural gene toachieve the enhancement.
 8. The process according to claim 1, wherein toachieve the enhancement, the life of the mRNA read off as the matrixfrom the above-mentioned sequences is prolonged and/or the breakdown ofthe corresponding enzyme protein(s) is prevented.
 9. The processaccording to claim 1, wherein the microorganism has additionalmetabolite or antimetabolite resistance mutations, individually ortogether.
 10. The process according to claim 1, wherein to achieve theover-expression the microorganisms are fermented in an appropriatelymodified culture medium or the fermentation procedure is modified. 11.The process according to claim 1, in which a) the D-pantothenic acidand/or the salt thereof is concentrated in the fermentation broth or inthe cells of the microorganism, and b) after the end of the fermentationthe desired product(s) is/are isolated, the biomass and/or furtherconstituents of the fermentation broth being separated off in an amountof ≧0 to 100%.
 12. The process according to claim 1, wherein themicroorganism of the Enterobacteriaceae family belongs to the genusEscherichia.
 13. The process according to claim 1, wherein themicroorganism is an Escherichia coli.
 14. The process according to claim1, wherein one or more of the genes selected from the group consistingof the following is or are additionally enhanced: the ilvGM operon whichcodes for acetohydroxy-acid synthase II, the panB gene which codes forketopantoate hydroxymethyl transferase, the panE gene which codes forketopantoate reductase, the pand gene which codes for aspartatedecarboxylase, the panC gene which codes for pantothenate synthetase,individually or together, the genes gcvT, gcvH and gcvP which code forthe glycine cleavage system, the glyA gene which codes for serinehydroxymethyl transferase, the serA gene which codes for phosphoglycericacid dehydrogenase, the serA(FBR) allele which codes for “feed back”resistant variants of phosphoglyceric acid dehydrogenase, the serC genewhich codes for phosphoserine transaminase, the bfr gene which codes forbacterioferrin, the hns gene which codes for the DNA-binding proteinHLP-II, the pgm gene which codes for phosphoglucomutase, the mdh genewhich codes for malate dehydrogenase, the cysK gene which codes forcysteine synthase A, the fda gene which codes for fructose bisphosphatealdolase (class II), the dldH gene which codes for NADH-dependentlipoamide dehydrogenase, the aldH gene which codes for NADP-dependentaldehyde dehydrogenase, and the adk gene which codes for adenylatekinase.
 15. The process according to claim 14, wherein said one or moreof the genes is or are overexpressed.
 16. The process according to claim1, wherein in the microorganism one or more genes which code for themetabolic pathways which reduce the formation of D-pantothenic acid areat least partly eliminated.
 17. The process according to claim 16,wherein said one or more genes which code for the metabolic pathwayswhich reduce the formation of D-pantothenic acid are avtA gene whichcodes for transaminase C, and/or the pckA gene which codes for PEPcarboxykinase.
 18. The process according to claim 1, wherein theexpression of the polynucleotide(s) which code(s) for the poxB gene isattenuated.
 19. The process according to claim 1, wherein the expressionof the polynucleotide(s) which code(s) for the poxB gene is eliminated.20. A process for the preparation of a feedstuffs additive comprisingD-pantothenic acid and/or a salt thereof comprising: a) separating thebiomass and/or a portion of the constituents in an amount of ≧0 to 100%from a fermentation broth obtained by fermenting at least onemicroorganism of the Enterobacteriaceae family, in which at least thenucleotide sequence(s) which code(s) for the pepB gene is/are enhanced,and comprising D-pantothenic acid, b) optionally, concentrating themixture from a), and c) converting the mixture into a finely dividedpowder to produce a a free-flowing animal feedstuffs additive with aparticle size distribution of 20 to 2000 μm.
 21. The process accordingto claim 20, wherein the particle size distribution is100 to 1400 μm.22. The process according to claim 20, wherein the animal feedstuffsadditive contains a salt of D-pantothenic acid selected from the groupconsisting of the magnesium and the calcium salt, wherein thefermentation of the microorganism is carried out in the presence ofcompounds of Ca or Mg, these being fed in continuously ordiscontinuously.
 23. The process according to claim 22, in whichstoichiometric amounts of the compounds of Ca or Mg are fed incontinuously or discontinuously.
 24. The process according to claim 20,wherein the animal feedstuffs additive contains a salt of D-pantothenicacid selected from the group consisting of the magnesium and the calciumsalt, wherein after the fermentation compounds of calcium or magnesiumare added to the fermentation broth, optionally after separating off ≧0to 100% of the biomass formed.
 25. The process according to claim 24,wherein the compounds of calcium or magnesium are added to thefermentation broth in stoichiometric amounts.
 26. The process accordingto claim 20, wherein before or after the concentration, D-pantothenicacid or one or more salts thereof is/are added to the fermentationbroth, the amount of compounds added being such that the totalconcentration thereof in the animal feedstuffs additive is in the rangefrom 20 to 80 wt. % (dry weight).
 27. The process according to claim 20,wherein the animal feedstuffs additive with the desired particle size isobtained from the fermentation broth, optionally after addition ofD-pantothenic acid and/or salts thereof, and optionally after additionof organic or inorganic auxiliaries, by a) drying and compacting, or b)spray drying, or c) spray drying and granulation, or d) spray drying andbuild-up granulation.
 28. The process according to claim 20, wherein thefermentation broth is applied to an inorganic auxiliary, optionallyafter removal of the biomass.
 29. The process according to claim 28,wherein the inorganic auxiliary is a silica or silicate.
 30. Amicroorganism of the Enterobacteriaceae family which producespantothenic acid and in which the pepB gene is present in enhanced form.